Patronin governs minus-end-out orientation of dendritic microtubules to promote dendrite pruning in Drosophila
- National University of Singapore, Singapore
Figures
Figure 1 with 1 supplement
Patronin is required for dendrite pruning in ddaC neurons.
(A) A schematic representation of dendrite pruning in ddaC neurons. (B–F) Live confocal images of ddaC neurons expressing mCD8-GFP driven by ppk-Gal4 at WP and 16 hr APF. While the wild-type neurons …
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Figure 1—source data 1
Extended statistical data as Microsoft Excel spreadsheet.
- https://doi.org/10.7554/eLife.39964.005
Figure 1—figure supplement 1
Patronin is required for dendrite pruning and arborization in sensory neurons.
(A–E) Live confocal images of da neurons expressing mCD8-GFP at w3L, WP, 16 hr and 20 hr, 32 hr APF. (A) While the control neurons pruned all the dendrites at 16 hr APF, ddaC neurons overexpressing p…
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Figure 1—figure supplement 1—source data 1
Extended statistical data as Microsoft Excel spreadsheet.
- https://doi.org/10.7554/eLife.39964.004
Figure 2 with 1 supplement
Overexpression of Patronin causes dendrite pruning defects in ddaC neurons.
(A–E) Live confocal images of ddaC neurons expressing mCD8-GFP driven by ppk-Gal4, Gal44-77 or ppk-CD4-tdGFP at WP and 16 hr APF. While ddaC neurons overexpressing the UAS control construct pruned …
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Figure 2—source data 1
Extended statistical data as Microsoft Excel spreadsheet.
- https://doi.org/10.7554/eLife.39964.009
Figure 2—figure supplement 1
Overexpression of Patronin causes dendrite arborization defects in ddaC neurons.
(A) Confocal images of ddaC neurons expressing mCD8-GFP and immunostained for anti-Patronin at w3L stage. ddaC somas are marked by dashed lines, and axons by arrows. Endogenous Patronin was detected …
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Figure 2—figure supplement 1—source data 1
Extended statistical data as Microsoft Excel spreadsheet.
- https://doi.org/10.7554/eLife.39964.008
Figure 3 with 1 supplement
The CKK domain is important for Patronin to govern dendrite pruning.
(A–D, G–J) Live confocal images of ddaC neurons expressing mCD8-GFP driven by ppk-Gal4 or Gal44-77 at WP and 16 hr APF. Low-level expressions of PatroninΔCH (B) and PatroninCKK (D), but not PatroninΔ…
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Figure 3—source data 1
Extended statistical data as Microsoft Excel spreadsheet.
- https://doi.org/10.7554/eLife.39964.013
Figure 3—figure supplement 1
Structure-function analysis of the Patronin protein.
(A) A schematic representation of full-length and truncated Patronin proteins and a summary of structure-function analysis of Patronin. Patronin contains multiple domains: a calponin homology (CH) …
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Figure 3—figure supplement 1—source data 1
Extended statistical data as Microsoft Excel spreadsheet.
- https://doi.org/10.7554/eLife.39964.012
Figure 4 with 1 supplement
Patronin is required for proper distribution of dendritic and axonal MT markers.
(A–C, F–H) Confocal images of ddaC neurons expressing mCD8-GFP, Nod-β-gal or Kin-β-gal and immunostained for β-galactosidase at wL3 stage. ddaC somas are marked by asterisks, axons by arrows and …
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Figure 4—source data 1
Extended statistical data as Microsoft Excel spreadsheet.
- https://doi.org/10.7554/eLife.39964.016
Figure 4—figure supplement 1
Patronin is required for proper distribution of the MT marker Nod-β-gal in ddaC dendrites.
(A) Confocal images of ddaC neurons expressing mCD8-GFP, Nod-β-gal and immunostained for β-galactosidase at wL3 stage. Nod-β-gal levels were reduced in the dendrites but enriched in the somas of patr…
Figure 5 with 1 supplement
Patronin is required for the minus-end-out orientation of dendritic MTs in ddaC neurons.
(A–B, G–H) Representative kymographs of EB1-GFP comets in control or mutant ddaC dendrites. In control RNAi (A) or UAS control (G) ddaC neurons, dendritic EB1-GFP comets predominantly moved …
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Figure 5—source data 1
Extended statistical data as Microsoft Excel spreadsheet.
- https://doi.org/10.7554/eLife.39964.020
Figure 5—figure supplement 1
Patronin is required for dendritic and axonal MT orientations in ddaC neurons.
(A) Representative kymographs of EB1-GFP comets in control or mutant ddaC dendrites. In control RNAi ddaC neurons, dendritic EB1-GFP comets predominantly moved retrogradely towards the somas. …
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Figure 5—figure supplement 1—source data 1
Extended statistical data as Microsoft Excel spreadsheet.
- https://doi.org/10.7554/eLife.39964.019
Figure 6 with 1 supplement
Attenuation of Klp10A, a kinesin-13 MT depolymerase, suppresses the patronin phenotypes in MT orientation in ddaC dendrites.
(A, B) Representative kymographs of EB1-GFP comets in control or mutant ddaC dendrites. In patronin RNAi #1 and control RNAi co-expressing ddaC neurons (A), dendritic EB1-GFP comets moved …
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Figure 6—source data 1
Extended statistical data as Microsoft Excel spreadsheet.
- https://doi.org/10.7554/eLife.39964.024
Figure 6—figure supplement 1
Attenuation of Klp10A, a kinesin-13 MT depolymerase, suppresses the patronin phenotype in MT orientation in ddaC dendrites.
(A) Quantification of the percentage of dendrite branches showing different levels of anterograde EB1-GFP comets in the control and mutant ddaC neurons. (B) Representative kymographs of EB1-GFP …
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Figure 6—figure supplement 1—source data 1
Extended statistical data as Microsoft Excel spreadsheet.
- https://doi.org/10.7554/eLife.39964.023
Figure 7 with 1 supplement
Patronin promotes dendrite pruning by orienting uniform minus-end-out MT arrays in dendrites.
(A–C, F–H) Live confocal images of ddaC neurons expressing mCD8-GFP driven by ppk-Gal4 at WP and 16 hr APF. Red arrowheads point to the ddaC somas. (A–C) ddaC neurons co-expressing patronin RNAi #1 …
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Figure 7—source data 1
Extended statistical data as Microsoft Excel spreadsheet.
- https://doi.org/10.7554/eLife.39964.028
Figure 7—figure supplement 1
patronin appears not to genetically interact with kat-60, kat-60L1 or tau in dendrite pruning.
(A–E) Live confocal images of ddaC neurons expressing mCD8-GFP driven by ppk-Gal4 at 16 hr APF. Similar to the control neurons, klp10A RNAi (#1 or #2) or kat-60 RNAi (#1 or #2) ddaC neurons pruned …
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Figure 7—figure supplement 1—source data 1
Extended statistical data as Microsoft Excel spreadsheet.
- https://doi.org/10.7554/eLife.39964.027
Figure 8 with 3 supplements
Klp10A overexpression phenocopies patronin knockdown in dendrite pruning and dendritic MT orientation.
(A–C) Live confocal images of ddaC neurons expressing mCD8-GFP driven by ppk-Gal4 at WP and 16 hr APF. Red arrowheads point to the ddaC somas. ddaC neurons overexpressing the UAS control construct …
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Figure 8—source data 1
Extended statistical data as Microsoft Excel spreadsheet.
- https://doi.org/10.7554/eLife.39964.036
Figure 8—figure supplement 1
Overexpression of Klp10A causes dendritic MT orientation defects in ddaC neurons.
(A–B) Confocal images of ddaC neurons expressing mCD8-GFP, Nod-lacZ or Kin-lacZ and immunostained for β-galactosidase at wL3 stage. ddaC somas are marked by asterisks, axons by arrows and dendrites …
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Figure 8—figure supplement 1—source data 1
Extended statistical data as Microsoft Excel spreadsheet.
- https://doi.org/10.7554/eLife.39964.031
Figure 8—figure supplement 2
The CKK domain of Patronin is important for dendrite pruning and dendritic MT orientation.
(A, D) Live confocal images of ddaC neurons expressing mCD8-GFP driven by ppk-Gal4 at 16 hr APF. Low-level expression of PatroninCKK under the control of UASp promoter significantly rescued the …
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Figure 8—figure supplement 2—source data 1
Extended statistical data as Microsoft Excel spreadsheet.
- https://doi.org/10.7554/eLife.39964.033
Figure 8—figure supplement 3
CNN and APC1/2 appear to be required for dendrite pruning of ddaC neurons.
(A–D) Live confocal images of ddaC neurons expressing mCD8-GFP driven by ppk-Gal4 at 16 hr APF. (A–B) While the control neurons pruned all the dendrites, cnnhk21 neurons (A), or APC2N175K, APC1Q8 …
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Figure 8—figure supplement 3—source data 1
Extended statistical data as Microsoft Excel spreadsheet.
- https://doi.org/10.7554/eLife.39964.035
Tables
Key resources table
| Reagent type | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (D. melangoaster) | UAS-MicalN-ter | other | (Terman et al., 2002) | |
| Genetic reagent (D. melangoaster) | SOP-flp (#42) | other | (Matsubara et al., 2011) | |
| Genetic reagent (D. melangoaster) | ppk-Gal4 on II and III chromosome | other | (Grueber et al., 2003) | |
| Genetic reagent (D. melangoaster) | UAS-Kin-β-gal | other | (Clark et al., 1997) | |
| Genetic reagent (D. melangoaster) | UAS-EB1-GFP | other | (Stone et al., 2008) | |
| Genetic reagent (D. melangoaster) | UASp-mCherry-Patronin | other | (Nashchekin et al., 2016) | |
| Genetic reagent (D. melangoaster) | patroninc9-c5 | other | (Nashchekin et al., 2016) | |
| Genetic reagent (D. melangoaster) | UAS-GFP-Patronin | other | (Derivery et al., 2015) | |
| Genetic reagent (D. melangoaster) | UASp-Arf79F-EGFP | other | (Shao et al., 2010) | |
| Genetic reagent (D. melangoaster) | UAS-Klp10A | this paper | ||
| Genetic reagent (D. melangoaster) | UAS-GFP-Klp10A | this paper | ||
| Genetic reagent (D. melangoaster) | UAS-Patronin | this paper | ||
| Genetic reagent (D. melangoaster) | UAS-Venus-Patronin | this paper | ||
| Genetic reagent (D. melangoaster) | UASp-PatroninΔCH | this paper | ||
| Genetic reagent (D. melangoaster) | UASp-PatroninΔCKK | this paper | ||
| Genetic reagent (D. melangoaster) | UASp-PatroninCKK | this paper | ||
| Genetic reagent (D. melangoaster) | UAS-Venus-PatroninΔCH | this paper | ||
| Genetic reagent (D. melangoaster) | UAS-Venus-PatroninΔCKK | this paper | ||
| Genetic reagent (D. melangoaster) | UAS-Venus-PatroninCC1-3 | this paper | ||
| Genetic reagent (D. melangoaster) | UAS-Venus-PatroninCKK | this paper | ||
| Genetic reagent (D. melangoaster) | UAS-Venus-PatroninCH | this paper | ||
| Genetic reagent (D. melangoaster) | Gal4109(2)80 | Bloomington Stock Center | BDSC: 8769 | |
| Genetic reagent (D. melangoaster) | ppk-CD4-tdGFP | Bloomington Stock Center | BDSC: 35843 | |
| Genetic reagent (D. melangoaster) | GSG2295-Gal4 | Bloomington Stock Center | BDSC: 40266 | |
| Genetic reagent (D. melangoaster) | Gal44-77 | Bloomington Stock Center | BDSC: 8737 | |
| Genetic reagent (D. melangoaster) | UAS-Nod-β-gal | Bloomington Stock Center | BDSC: 9912 | |
| Genetic reagent (D. melangoaster) | patronin RNAi #2 | Bloomington Stock Center | BDSC: 36659 | |
| Genetic reagent (D. melangoaster) | klp10A RNAi #2 | Bloomington Stock Center | BDSC: 33963 | |
| Genetic reagent (D. melangoaster) | Gal42-21 | Bloomington Stock Center | FBal0328157 | |
| Genetic reagent (D. melangoaster) | patronin RNAi # 1 | Vienna Drosophila RNAi Centre | VDRC: v108927 | |
| Genetic reagent (D. melangoaster) | klp10A RNAi # 1 | Vienna Drosophila RNAi Centre | VDRC: v41534 | |
| Genetic reagent (D. melangoaster) | control RNAi | Vienna Drosophila RNAi Centre | VDRC: v36355 | |
| Genetic reagent (D. melangoaster) | control RNAi | Vienna Drosophila RNAi Centre | VDRC: v37288 | |
| Genetic reagent (D. melangoaster) | patronink07433 | Drosophila Genetic Resource Center | #111217 | |
| Genetic reagent (D. melangoaster) | patronin RNAi #3 | National Institute of Genetics, Japan | #18462 Ra-1 | |
| Genetic reagent (D. melangoaster) | kat-60 RNAi # 1 | Vienna Drosophila RNAi Centre | VDRC: v38368 | |
| Genetic reagent (D. melangoaster) | kat-60 RNAi # 2 | Vienna Drosophila RNAi Centre | VDRC: v106487 | |
| Genetic reagent (D. melangoaster) | kat-60L1 RNAi # 1 | Vienna Drosophila RNAi Centre | VDRC: v31599 | |
| Genetic reagent (D. melangoaster) | kat60-L1 RNAi # 2 | Vienna Drosophila RNAi Centre | VDRC: v108168 | |
| Genetic reagent (D. melangoaster) | tau RNAi # 1 | Bloomington Stock Center | BDSC: 28891 | |
| Genetic reagent (D. melangoaster) | tau RNAi # 2 | Bloomington Stock Center | BDSC: 40875 | |
| Genetic reagent (D. melangoaster) | cnnhk21 | Bloomington Stock Center | BDSC: 5039 | |
| Genetic reagent (D. melangoaster) | FRT 82B, APC2N175K, APC1Q8 | Bloomington Stock Center | BDSC: 7211 | |
| Genetic reagent (D. melangoaster) | futschN94 | Bloomington Stock Center | BDSC: 8805 | |
| Antibody | anti-β-galactosidase | Promega | Cat#: Z3783 | 1:1000 |
| Antibody | anti-Patronin | M Gonzalez-Gaitan | 1:500 | |
| Antibody | Cy3-conjugated goat anti-Rabbit antibody | Jackson | Cat#111-165-003 | 1:500 |
| Antibody | 647-conjugated goat anti-Rabbit antibody | Jackson | Cat#111-605-144 | 1:500 |
| Antibody | anti-Futsch | DSHB | 22c10 | 1:50 |
Additional files
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Supplementary file 1
Supplementary list of fly strains.
- https://doi.org/10.7554/eLife.39964.037
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Transparent reporting form
- https://doi.org/10.7554/eLife.39964.038
